Cytokine Immunoassays: New Methods to Evaluate Steller
Sea Lion Immune Health
Mary Bozza, Research AssociateMary Bozza, Research Associate
Alaska SeaLife CenterAlaska SeaLife Center
Steller Sea Lion Immune Health
• Do Steller sea lions bioaccumulate contaminants to toxic levels?
• How can we measure toxicity?• Toxins can cause immune system disruption• Marine contaminants linked to
immunosuppression include: Organochlorines, PCB’s, Cadmium
Steller Sea Lion Immune Health
Alaska SeaLife Center Contaminants Project: Endocrinology, Immunology and ToxicologyS. Aktkinson, B. Middlebrooks, Q. Li
Individual Health Assessment of Steller Sea Lions in AlaskaBurek, Beckmen, Rea, and Gellat
Project Goals:
I. Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitro
II. Evaluate immunocompetence:• Establish baseline cytokine profiles to assess
immune health of individual Steller sea lions
• Correlate cytokine profiles with
• immune and overall health parameters
• body burdens of contaminants to determine toxicity
What are Cytokines?
• Proteins secreted by immune cells in response to inflammation or other stress
• Called “interleukins,” they act as chemical messengers to regulate inflammation
• Routinely used to study other mammalian immune systems including human & mouse
• Commercially available reagents may be available to measure Steller sea lion cytokines if appropriate assays are developed
An immunoassay uses an antibody to recognize the protein of interest; the concentration of the antibody-protein complex is measured
Challenge:• Antibodies to Steller sea lion cytokines do not exist!• Proteins are not available to generate or validate
antibodies
Solution:• Make Steller sea lion protein using molecular cloning
techniques• Use the protein to validate existing cross-reactive
antibodies to develop an assay
Project Goals:
I. Develop assays to measure Steller sea lion cytokines in serum and from white blood cells in vitro
II. Evaluate immunocompetence:
• Establish baseline cytokine profiles to assess immune health of individual Steller sea lions
• Correlate cytokine profiles with
• immune and overall health parameters
• body burdens of contaminants to determine toxicity
6. Validate assay using cross-reactive antibodies
1. SSL immune cells cultured in vitro with a bacterial extract
(LPS)
2. Isolate mRNA
4. Clone and sequence full-length cytokine
genes by PCR
3. Construct a cDNA library
5. Protein Expression
Goal I: Develop Assays
Step 1. LPS Stimulation of Mononuclear Cells
Collect whole blood
Place cells in culture dish, add LPS
(lipopolysaccharide) to stimulate immune
response
Centrifuge blood over a Ficoll-
Hypaque gradient to isolate mononuclear
cells
Cell Signaling
TranscriptionStep 2. Isolate
mRNA
Translation Secretion
Step 2: Isolate mRNA
Size fractionation by electrophoresis (1% agarose gel)
1. Total RNA from HS LPS stimulated mononuclear cells
2. PolyA+ Control RNA (human placenta)
1 2
Step 3. Construct a cDNA Library
• Isolate mRNA transcripts from tissues or cells. • Convert transcripts into double-stranded DNA
fragments called cDNA.
Chromosome Genomic DNA mRNA cDNA
• Insert cDNA into plasmid vectors that can replicate in bacteria.• Introduce vectors into bacteria.• Amplify library.
cDNA
cDNA inserted into plasmid
plasmid in bacteria
amplified library
Advantages of a cDNA Library
• Contains only protein-coding DNA (genes)• Represents the entire collection of genes expressed in
cell or tissue type• Vector with inserted gene can be used to produce
protein• Can amplify and store the library for long term use• Clone many other genes from stimulated white blood
cell cDNA library• New libraries from Steller sea lion cells or cell lines• New libraries from other species cells or cell lines
6. Validate assay using cross-reactive antibodies
1. SSL mononuclear cells cultured in vitro with a bacterial extract (LPS)
2. Isolate mRNA
4. Clone and sequence full-length cytokine
genes by PCR
3. Construct a cDNA library
5. Protein Expression
Step 7. Assay Development
• ELISA (enzyme linked immunosorbent assay) or RIA (radioimmunoassay)
• Test commercially available antibodies for binding to protein
• Validate assay using purified protein
• Validate assay using serum
Ways to Use New AssaysCollect blood
samples
Cytokine ELISAs(Inflammation,
Infection or Disease)
Stimulate WBC in vitro
(Immunocompetence)
Cytokine ELISAs
White blood cells
Serum
Ways to Use New AssaysCollect blood
samples
Cytokine Profiles by
RT-PCR(Inflammation,
Infection or Disease)
Cytokine ELISAs(Inflammation,
Infection or Disease)
Stimulate WBC in vitro
(Immunocompetence)
Cytokine Profiles by RT-PCR
Cytokine ELISAs
White blood cells
Serum
Steller Sea Lion Immune Health
• Do Steller sea lions bioaccumulate contaminants to toxic levels?
• How can we measure toxicity?• Toxins can cause immune system disruption• Marine contaminants linked to
immunosuppression include: Organochlorines, PCB’s, Cadmium
What’s next?
• LPS stimulation done• cDNA library is being
constructed• Next step:
• PCR Cloning
• Collaboration• Vectors, methods for
protein production
• Which genes to clone, proteins to assay?
• Additional libraries?
Acknowledgements
• Dr. Shannon Atkinson• Don Calkins• Dr. Jo-Ann Mellish• ASLC Mammal Dept.• ASLC Veterinary Services Dept.• Carol Stephens• Blood Donors: Woody, Kiska,
Sugar, Tina and Pender• Jon Moreland• Lizabeth Moundalexis• Annette D’Alessandro & Angie Steeves
